This invention relates to an immobilized enzyme membrane, a process for preparing the membrane, and an enzyme electrode apparatus for electrochemically measuring instruments using the immobilized enzyme membrane.
Heretofore, various analytical methods using an immobilized enzyme membrane have been proposed for quantitatively analyzing medically important substances in living bodies, for example, sacharides, urea, chloesterol, or other substances contained in very small amounts in the living body liquids with a good selectivity. These methods provide effective means for detecting components contained in very small amounts in a multi-component liquid by utilizing a substrate peculiarity and a high catalytic activity possessed by the enzyme. However, these methods still have a problem in immobilizing the enzyme and have not been yet widely practically utilized.
The enzyme has been so far immobilized in a membrane state or a membrane according to any of the following methods: (1) a method of enclosing the enzyme with a polyacrylamide gel [Nature 214 986 (1967)], (2) a method of mixing the enzyme with an inert protein such as albumin, etc. as a reinforcing agent, and cross-linking the inert protein by a cross-linking agent [Biotechnology and Bioengineering 15 359 (1973)], (3) a method of absorbing the enzyme in filter paper or cellophane, and cross-linking it by glutaraldehyde [Biotechnology and Bioengineering 15 359 (1973)], (4) a method of ionically bonding the enzyme with an ion-exchange cellulose [Biotechnology and Bioengineering 13 (1971)], (5) a method of adding the enzyme to a collagen fiber solution, placing the solution in an electrolysis cell, passing an electric current through the cell, and electro-depositing a collagen film enclosing the enzyme onto an electrode [Biochemistry, Biophysics Research Communication 47 51 (1972)], (6) a method of physico-chemically immobilizing the enzyme on a porous, organopolymeric film (Japanese Laid-open Patent Application Specification No. 17,889/77), (7) a method of sandwiching an enzyme gel in between two films (Japanese Laid-open Patent Application Specification No. 55,691/77), etc.
A large amount of enzyme can be immobilized according to said method (1), but the strength of a membrane is not enough, and diffusion of substrates and products is poor. According to said method (2), an enzyme load can be increased, but the strength is not sufficient, and a resistance to microorganisms is not sufficient because the protein is used as the reinforcing agent. Said method (3) can be easily carried out, but the enzyme load is not sufficient, and when a film is made thicker to increase the strength, diffusion of substrate, etc. becomes poor; whereas when the film is made thinner, the strength becomes poor. Both methods (4) and (5) can be easily carried out, but a bonding of the enzyme to a carrier is weak, and the enzyme is easily released from the carrier. Said method (6) is to overcome the foregoing drawbacks, but the membrane is perforated from the face side to the back side of the membrane, and thus a sufficient amount of the enzyme cannot be retained in the perforations of the porous membrane. Furthermore, the membrane has not a sufficient porosity and thus the enzyme load is not sufficient. Said method (7) is complicated in its preparation, because the enzyme gel is sandwiched in between two thin films, and increases the production cost, though its enzyme load is large.